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How do Plants Make Oxygen? Ask Cyanobacteria

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Researchers reveal the family tree of the group of microorganisms responsible for "inventing" the oxygen-producing photosynthesis that lets you breathe
News Writer: 
Robert Perkins
Bacterial mats
Oxyphotobacteria in microbial mats in Yellowstone.
Credit: Fischer Laboratory/Caltech

The ability to generate oxygen through photosynthesis—that helpful service performed by plants and algae, making life possible for humans and animals on Earth—evolved just once, roughly 2.3 billion years ago, in certain types of cyanobacteria. This planet-changing biological invention has never been duplicated, as far as anyone can tell. Instead, according to endosymbiotic theory, all the "green" oxygen-producing organisms (plants and algae) simply subsumed cyanobacteria as organelles in their cells at some point during their evolution.

"Oxygenic photosynthesis was an evolutionary singularity," says Woodward Fischer, professor of geobiology at Caltech, referring to the process by which certain organisms use the energy of sunlight to convert carbon dioxide and water into sugar for food, with oxygen as a by-product. "Cyanobacteria invented it, and then ultimately become the chloroplasts of algae. Plants are just a group of algae that moved on land."

Yet as world-shaping as cyanobacteria are, relatively little is known about them. Until a couple of decades ago, they were called "blue-green algae" by taxonomists, though it was later revealed that they are not algae at all, but rather a completely different type of organism. That lack of taxonomic understanding made deciphering the riddle of their evolution all but impossible, Fischer says.

"For the longest time, they were just their own group. We had no answer about where they came from, or what other organisms they were related to," Fischer says. "Imagine trying to understand something about human evolution without knowledge of the great apes."

Publishing in the journal Science on March 30, Fischer and colleagues from Caltech and the University of Queensland in Australia finally have fleshed out cyanobacteria's family tree. They added the genomes of 41 uncultured microorganisms, which helped to pin down the precise point in the evolution of cyanobacteria at which oxygenic photosynthesis arose. The 41 species are all types of cyanobacteria but none carry genes for photosynthesis, and therefore they don't produce organic matter, like algae and plants do. Rather, they consume it.

Fischer and his colleagues found that a single branch of cyanobacteria—dubbed Oxyphobacteria—were likely the first and only group to evolve oxygenic photosynthesis. Their closest relatives, Melainabacteria, live in the guts of animals (including humans) among other environments, and do not produce oxygen. And while one might suggest that Melainabacteria simply lost the ability to produce oxygen over time, the next most closely related cyanobacteria after those, described in the paper as Sericytochromatia, also do not engage in oxygenic photosynthesis.

"This nails down that Oxyphobacteria were really the only ones to ever invent this globe-shaping chemical process," Fischer says.

The 41 new species fall into both Melainabacteria and Sericytochromatia, the latter of which had not been described before this paper. All names of these organisms are subject to change, as taxonomists catch up with the team's discoveries. "We know they're there, and we know their gene repertoire. Now we can start putting them into evolutionary trees, and begin efforts to isolate them and study their physiology and ecology," says James Hemp, an Agouron Postdoctoral Scholar at Caltech when the research was conducted, and coauthor of the Science article.

These discoveries were made thanks to new technology that allows researchers to sequence the genome of an organism without first having to isolate that organism in the lab and culture a large quantity of it, as has been required in the past. 

"Now we have culture-independent ways of assessing microbial diversity," says Rochelle M. Soo, postdoctoral researcher at the University of Queensland in Australia and coauthor of the Science article. "We can go into any environment, remove a sample of DNA, sequence it, and recover genomes of microbes living in that environment. We don't have to grow anything ourselves—instead we let the environment do the work and just sequence what's already there." Some of the 41 new species were found in situ, such as in the intestines of animals, while others came from the databases of other biology studies—which had been sampled, but never characterized and analyzed. 

Unraveling the evolutionary mystery of photosynthesis and its genesis could shed light on everything from sustainable energy sources to the potential for life to exist on other planets.

"Cyanobacteria are planetary-scale engineers, capable of splitting water. They invented the most challenging chemistry on the face of the planet. We would love to be able to do their water-splitting chemistry as effortlessly as they do to make fuels, and these guys figured out how to do it two and a half billion years ago," Fischer says.

Next, the team plans to learn more about the ecology and physiology of the new bacteria by probing them in a lab. "We've really just scratched the surface," Fischer says.

The study is titled "On the origins of oxygenic photosynthesis and aerobic respiration in Cyanobacteria." Coauthors include Donovan H. Parks and Philip Hugenholtz of the University of Queensland in Australia. This research was funded by a Discovery Outstanding Researcher Award, the Australian Research Council, the Agouron Institute, NASA, and the David and Lucile Packard Foundation. Sequencing data have been deposited at the National Center for Biotechnology Information. 


Scientists Learn Secrets of Deadly Bacterial Toxin Gun

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First 3-D image reveals previously unknown details about the bacterial machine that injects toxins into cells and spreads antibiotic resistance
News Writer: 
Lori Dajose
Animated gif of an artist's rendition of a cellular machine spewing toxic molecules.
Artist's depiction of a type IV secretion system nestled within a bacterial cell membrane. This structure shoots out thousands of toxic molecules (purple) into human cells during an infection.
Credit: Caltech

Experts predict that by 2050, antibiotic-resistant bacteria will cause as many deaths as cancer. Now, for the first time, Caltech scientists have created a 3-D image of a molecular structure that many different bacteria use to pump toxins into human cells and spread antibiotic-resistance genes to other bacteria. Understanding the architecture of this structure is a first step toward combating its effects.

The study was conducted in the laboratory of Grant Jensen, professor of biophysics and biology and Howard Hughes Medical Institute Investigator. A paper describing the work first appeared online in the March 23 issue of EMBO Reports.

The researchers looked specifically at Legionella, the bacteria that causes Legionnaires' disease, a severe and often lethal form of pneumonia. When Legionella invades a human cell, it wraps itself in a protective vesicle and opens the molecular structure, known as a type IV secretion system. The molecular "machine" sits in the cell membrane of the bacterium and proceeds to shoot tens of thousands of toxic molecules—hundreds of different types—into the human cell, hijacking cellular pathways and overwhelming the cell's defenses.

Some type IV secretion systems are thought to be instrumental in spreading antibiotic-resistance genes throughout the bacterial population.

"Understanding the structure of the type IV system is crucial to developing new antibiotics to disable it," says first author and postdoctoral scholar Debnath Ghosal. "While this study focuses only on the secretion system of Legionella, a very similar machine is used by many bacteria—such as the pathogens that cause stomach cancer, Q fever, and whooping cough."

To image the structure—which, at 40 nanometers in diameter, is about 1,000 times too small to be seen by the human eye—the researchers employed a technique called electron cryotomography. In this method, bacteria are frozen alive and then rotated under a powerful electron microscope to create a series of 2-D images that can be digitally reconstructed into a 3-D picture. This was the first-ever image of a type IV machine within a bacterium.

The imaging revealed that the structure is shaped into concentric arches, like the symbol for Wi-Fi. Understanding the structure should eventually aid efforts to design drugs that can block the machine. Developing a drug that would disable even one core protein component of the secretion system, Ghosal says, would enable human cells to fight back against the bacterial infection.

"Most current antibiotics focus on destroying the cellular envelope that encompasses a bacterial cell, preventing it from replicating," says Jensen. "Developing new antibiotics that target different aspects of the bacterial cell, such as the type IV secretion system, would enable us to block infections in additional ways."

The paper is titled "In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography." In addition to Ghosal and Jensen, coauthors are Caltech research scientist Yi Wei Chang, Kwang Cheol Jeong of the Washington University School of Medicine and the University of Florida, and Joseph Vogel of the Washington University School of Medicine. Funding was provided by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases.

Bare Bones: Making Bones Transparent

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Transparent bones enable researchers to observe the stem cells inside
News Writer: 
Lori Dajose
A bone rendered transparent. Stem cells are colored red.
A mouse tibia that has been rendered transparent with Bone CLARITY. Stem cells appear distributed throughout the bone in red. The ability to see bone stem cell behavior is crucial for testing new osteoporosis treatments.
Credit: Science Translational Medicine, Greenbaum, Chan, et al; Gradinaru laboratory/Caltech

Ten years ago, the bones currently in your body did not actually exist. Like skin, bone is constantly renewing itself, shedding old tissue and growing it anew from stem cells in the bone marrow. Now, a new technique developed at Caltech can render intact bones transparent, allowing researchers to observe these stem cells within their environment. The method is a breakthrough for testing new drugs to combat diseases like osteoporosis.

The research was done in the laboratory of Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering and a Heritage Medical Research Institute Investigator. It appears in a paper in the April 26 issue of Science Translational Medicine.

In healthy bone, a delicate balance exists between the cells that build bone mass and the cells that break down old bone in a continual remodeling cycle. This process is partially controlled by stem cells in bone marrow, called osteoprogenitors, that develop into osteoblasts or osteocytes, which regulate and maintain the skeleton. To better understand diseases like osteoporosis, which occurs when loss of bone mass leads to a high risk of fractures, it is crucial to study the behavior of stem cells in bone marrow. However, this population is rare and not distributed uniformly throughout the bone.

"Because of the sparsity of the stem cell population in the bone, it is challenging to extrapolate their numbers and positions from just a few slices of bone," says Alon Greenbaum, postdoctoral scholar in biology and biological engineering and co-first author on the paper. "Additionally, slicing into bone causes deterioration and loses the complex and three-dimensional environment of the stem cell inside the bone. So there is a need to see inside intact tissue."

To do this, the team built upon a technique called CLARITY, originally developed for clearing brain tissue during Gradinaru's postgraduate work at Stanford University. CLARITY renders soft tissues, such as brain, transparent by removing opaque molecules called lipids from cells while also providing structural support by an infusion of a clear hydrogel mesh. Gradinaru's group at Caltech later expanded the method to make all of the soft tissue in a mouse's body transparent. The team next set out to develop a way to clear hard tissues, like the bone that makes up our skeleton.

In the work described in the new paper, the team began with bones taken from postmortem transgenic mice. These mice were genetically engineered to have their stem cells fluoresce red so that they could be easily imaged. The team examined the femur and tibia, as well as the bones of the vertebral column; each of the samples was about a few centimeters long. First, the researchers removed calcium from the bones: calcium contributes to opacity, and bone tissue has a much higher amount of calcium than soft tissues. Next, because lipids also provide tissues with structure, the team infused the bone with a hydrogel that locked cellular components like proteins and nucleic acids into place and preserved the architecture of the samples. Finally, a gentle detergent was flowed throughout the bone to wash away the lipids, leaving the bone transparent to the eye. For imaging the cleared bones, the team built a custom light- sheet microscope for fast and high-resolution visualization that would not damage the fluorescent signal. The cleared bones revealed a constellation of red fluorescing stem cells inside.

The group collaborated with researchers at the biotechnology company Amgen to use the method, named Bone CLARITY, to test a new drug developed for treating osteoporosis, which affects millions of Americans per year.

"Our collaborators at Amgen sent us a new therapeutic that increases bone mass," says Ken Chan, graduate student and co-first author of the paper. "However, the effect of these therapeutics on the stem cell population was unclear. We reasoned that they might be increasing the proliferation of stem cells." To test this, the researchers gave one group of mice the treatment and, using Bone CLARITY, compared their vertebral columns with bones from a control group of animals that did not get the drug. "We saw that indeed there was an increase in stem cells with this drug," he says. "Monitoring stem cell responses to these kinds of drugs is crucial because early increases in proliferation are expected while new bone is being built, but long-term proliferation can lead to cancer."

The technique has promising applications for understanding how bones interact with the rest of the body.

"Biologists are beginning to discover that bones are not just structural supports," says Gradinaru, who also serves as the director of the Center for Molecular and Cellular Neuroscience at the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. "For example, hormones from bone send the brain signals to regulate appetite, and studying the interface between the skull and the brain is a vital part of neuroscience. It is our hope that Bone CLARITY will help break new ground in understanding the inner workings of these important organs."

The paper is titled "Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow." Other Caltech coauthors include incoming graduate students Tatyana Dobreva and David Brown. Helen J. McBride and Rogely Boyce of Amgen also coauthored the paper. Funding was provided by the National Institutes of Health, the Presidential Early Career Award for Scientists and Engineers, the Heritage Medical Research Institute, the Shurl & Kay Curci Foundation, the Amgen Chem-Bio-Engineering Awards, the Pew Charitable Trusts, the Kimmel Foundation, and the Caltech-City of Hope Biomedical Research Initiative. Alon Greenbaum is a Good Ventures Fellow of the Life Sciences Research Foundation.

Cells Calculate Ratios to Control Gene Expression

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Cells transmit signals in ratios
News Writer: 
Lori Dajose
cells moving
Behavior of cells over time after they have received a Tgf-beta signal. The brightness of the nuclei (circled in red) indicates how much Smad protein is present. This brightness varies from cell to cell, but the ratio of brightness after the signal to before the signal is about the same. The ratio guides the cell to modify gene expression accordingly, suggesting that cells can monitor protein abundance over time, and perform simple division to decipher protein abundance into gene expression.
Credit: Courtesy of the Goentoro lab

In multicellular animals, cells communicate by emitting and receiving proteins, a process called signaling. One of the most common signaling pathways is the transforming growth factor b (Tgf-b) pathway, which functions in all animal species throughout their lifetimes and regulates numerous biological processes, such as instructing cells how to differentiate—whether a stem cell will become a muscle cell or a bone cell, for example.

But how do cells decipher those signals and use that information to guide gene expression? The answer, according to new research from the laboratory of Lea Goentoro, assistant professor of biology at Caltech: the cells perform simple division. In other words, they do math.

The findings, which appear in a paper in the April 4 issue of Proceedings of the National Academy of Sciences, contradict previous hypotheses about how cells interpret signals. The work was led by Christopher Frick, a graduate student in biochemistry and molecular biophysics.

"Malfunctions in the Tgf-b pathway have been implicated in many types of cancer, and that's why it's important for us to study how it works, to understand how cells use this pathway to take information from their environment and turn it into altered gene expression," Goentoro says. "We found that cells are able to monitor the abundance of a certain protein over time and somehow divide the abundance after the Tgf-b signal by the abundance before the signal. The cell then uses this ratio to adjust gene expression accordingly."

The cell's detection of the Tgf-b signal triggers a series of molecular interactions, culminating in changes in the abundance of a protein called Smad3. A kind of messenger, Smad3 is activated at the cell membrane when a cell encounters the Tgf-β signal, and ultimately ends up in the nucleus of the cell where it directs gene expression. Biologists have commonly believed that the degree to which gene expression is changed will depend on how much Smad protein is produced after exposure to Tgf-β.

In order to study how Smad responds to Tgf-b in real time, the researchers made movies of the signaling process in individual cells. They discovered that the pathway did not behave as biologists had previously thought. After exposure to the Tgf-b signal, the researchers found, Smad3 did indeed move to the nucleus, as expected. The surprise was that the abundance of Smad3 was significantly different in each cell. And yet, despite those wildly varying concentrations of Smad3, the level of Smad3 after the signal divided by the level before the signal was consistent in each cell.

This observation led the researchers to hypothesize that cells are somehow able to compute this ratio and that the gene response is proportional to this relative change (called a fold-change) rather than to the abundance of Smad3.

To test this, the researchers measured target gene expression in cells and compared how it correlated with both the abundance of Smad3 as well as the fold-change of Smad3. Indeed, they found that the target gene expression depended only on the ratio of Smad3 and not on how much Smad3 a cell had.

"Prior to this work, researchers trying to characterize the properties of a tumor might take a slice from it and measure the total amount of Smad in cells," says Goentoro. "Our results show that to understand these cells one must instead measure the change in Smad over time."

"This result is nonintuitive: it is easy to imagine how a cell can increase the expression of a target gene by increasing the abundance of Smad3," says Frick. "Thus, many of us had expected that the abundance of Smad3 would be tuned in response to the Tgf-β signal. In contrast, what these cells regulate is the ratio of the change in abundance of Smad3. The big question now is: How does a cell compute this ratio and then adjust gene expression accordingly?"

The paper is titled "Sensing relative signal in the Tgf-β/Smad pathway." Other Caltech co-authors include former research technician Clare Yarka and graduate student Harry Nunns. Funding was provided by the National Institutes of Health, the James S. McDonnell Scholar Award in Complex Systems, and the National Science Foundation.

Taking a Closer Look at Genetic Switches in Cancer

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New research shows how a protein controls blood cell production in leukemia
News Writer: 
Whitney Clavin
Diagram of DPF2 in cell
The DPF2 protein controls a genetic switch that tells blood stem cells when to become red and white blood cells. When DPF2 is bound to DNA-protein complexes called nucleosomes at specific regions in the genome, it blocks the activation of genes that promote red and white blood cell production. When DPF2 is not bound, the switch is flipped on and blood cells are made. In some patients with acute myeloid leukemia, DPF2 is overproduced, blocking cell differentiation and indicating a poor prognosis.
Credit: Hoelz group/Caltech

Many things go wrong in cells during the development of cancer. At the heart of the chaos are often genetic switches that control the production of new cells. In a particularly aggressive form of leukemia, called acute myeloid leukemia, a genetic switch that regulates the maturation of blood stem cells into red and white blood cells goes awry. Normally, this switch leads to appropriate numbers of white and red blood cells. But patients with acute myeloid leukemia end up with a dangerous accumulation of blood stem cells and a lack of red and white blood cells—cells that are needed to supply the body with oxygen and fight infections.

Now, researchers at Caltech and the Sylvester Comprehensive Cancer Center at the University of Miami are narrowing in on a protein that helps control this genetic switch. In healthy individuals, the protein, called DPF2, stops the production of red and white blood cells when they do not need to be replaced. That is, it turns the switch off. But the protein can be overproduced in acute myeloid leukemia patients. The protein basically sits on the switch, preventing it from turning back on to make the blood cells as needed. Patients who overproduce DPF2 have a particularly poor prognosis.

In a new study, to be published the week of May 22, 2017, in the journal Proceedings of the National Academy of Sciences, the researchers demonstrate new ways to impede DPF2, potentially rendering acute myeloid leukemia more treatable. They report new structural and functional details about a fragment of DPF2. This new information reveals targets for the development of drugs that would block the protein's function.

"Many human diseases, including cancers, arise because of malfunctioning genetic switches," says André Hoelz, the corresponding author of the study. Hoelz is a professor of chemistry at Caltech, a Heritage Medical Research Institute (HMRI) Investigator, and a Howard Hughes Medical Institute (HHMI) Faculty Scholar. "Elucidating how they work at atomic detail allows us to begin the process of custom tailoring drugs to inactivate them and in many cases that is a significant step towards a cure."


Crystal structure of a portion of human DPF2, a protein that controls a genetic switch that tells blood stem cells when to become red and white blood cells. Orange and yellow regions illustrate the DPF2 "reader" domain, which is stabilized by zinc ions, represented as red and grey spheres. Credit: Hoelz Lab/Caltech

Red and white blood cells are constantly regenerated from blood stem cells, which reside in our bone marrow. Like other stem cells, blood stem cells can live forever. It is only when they become differentiated into specific cell types, such as red and white blood cells, that they then become mortal, or acquire the ability to die after a certain period of time.

"Our bodies use a complex series of genetic switches to differentiate a blood stem cell into many different cell types. These differentiated cells then circulate in the blood and serve a variety of different functions. When these cells reach the end of their lifespan they need to be replaced," says Hoelz. "This is somewhat like replacing used tires on a car."

To investigate the role of DPF2 and learn more about how it controls the genetic switch for making blood cells, the Hoelz group partnered with Stephen D. Nimer, co-corresponding author of the paper and director of the Sylvester Comprehensive Cancer Center, and his team. First, Ferdinand Huber and Andrew Davenport—both graduate students at Caltech in the Hoelz group and co-first-authors of the new study—obtained crystals of a portion of the DPF2 protein containing a domain known as a PHD finger, which stands for planet homeodomain. They then used X-ray crystallography, a process that involves exposing protein crystals to high-energy X-rays, to solve the structure of the PHD finger domain. The technique was performed at the Stanford Synchrotron Radiation Lightsource, using a dedicated beamline of Caltech's Molecular Observatory.

The results revealed how DPF2 binds to a DNA-protein complex, called the nucleosome, to block the production of red and white blood cells. The protein "reads" various signals displayed on the nucleosome surface by adopting a shape that fits various modifications on the nucleosome complex, like the different shaped pieces of a jigsaw puzzle. Once the protein binds to this DNA locus, DPF2 turns off the switch that regulates blood cell differentiation.

The next step was to see if DPF2 could be blocked in human blood stem cells in the lab. Sarah Greenblatt, a postdoctoral associate in Nimer's group and co-first author of the study, used the structural information from Hoelz's group to create a mutated version of the protein. The Nimer group then introduced the mutated protein in blood stem cells, and found that the mutated DPF2 could no longer bind to the nucleosome. In other words, DPF2 could no longer inactivate the switch for making blood cells.

"The mutated DPF2 was unable to bind to specific regions in the genome and could not halt blood stem cell differentiation," says Huber. "Whether DPF2 can also be blocked in the cancer patients themselves remains to be seen." The researchers say a structural socket in DPF2, one of the puzzle-piece-like regions identified in the new study, is a good target for candidate drugs.

The study, titled "Histone-Binding of DPF2 Mediates Its Repressive Role in Myeloid Differentiation," was funded by a PhD fellowship of the Boehringer Ingelheim Fonds, a National Institutes of Health Research Service Award, the National Cancer Institute of the National Institutes of Health, a Faculty Scholar Award of the Howard Hughes Medical Research Institute, the Heritage Medical Research Institute, Caltech startup funds, the Albert Wyrick V Scholar Award of the V Foundation for Cancer Research, a Kimmel Scholar Award of the Sidney Kimmel Foundation for Cancer Research, and a Teacher–Scholar Award of the Camille & Henry Dreyfus Foundation. Other authors are Concepcion Martinez and Ye Xu of the University of Miami and Ly P. Vu of the Memorial Sloan Kettering Cancer Center.

Sour Taste Cells Detect Water

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Sour-sensing taste pathway also mediates water detection in mammalian tongue
News Writer: 
Lori Dajose
Water pouring into a glass with a lemon in it.
Sour taste cells may not behave as biologists previously believed.
Credit: Oka Laboratory/Caltech

New research from Caltech shows that sour-sensing taste cells play an important role in detecting water on the tongue.

The work, appearing in a paper in the May 29 issue of the journal Nature Neuroscience, was done in the laboratory of Yuki Oka, assistant professor of biology.

"The tongue can detect various key nutrient factors, called tastants— such as sodium, sugar, and amino acids—through taste," says Oka. "However, how we sense water in the mouth was unknown. Many insect species are known to 'taste' water, so we imagined that mammals also might have a machinery in the taste system for water detection."

Taste cells relay information about tastants to the brain via nerves called the taste nerves. First author and graduate student Dhruv Zocchi measured the electrical responses from taste nerves in mice to various tastants as well as to water. The nerves responded in predictable ways to different basic tastes—sweet, sour, bitter, salty, and umami—but they were also stimulated by pure water. "This was exciting because it implied that some taste cells are capable of detecting water," Zocchi says.

Each basic taste is mediated by distinct subsets of taste cells. In order to test which taste cells respond to water, the team genetically and pharmacologically blocked the function of individual cell populations. For example, when the salt taste receptor was blocked, salt no longer triggered activity in taste nerves, but responses to other tastes were not affected. "To our surprise, when we silenced sour taste cells, water responses were also completely blocked," Oka says. "The results suggested that water is sensed through sour taste cells."

To prove that the sour cells indeed contribute to water detection, the team used a technique called optogenetics that allowed them to stimulate sour cells with light instead of water. The researchers removed water from the animals' water bottle and made it so that the bottle's spout emitted a blue light when the animals touched it. They discovered that thirsty genetically engineered mice would go to the spout for water, encounter the light, and "drink" it. Though the mice were not rehydrated, they kept licking the water source because the light created a sensory cue of water.

A sour taste is often associated with an unpleasant taste quality that reduces animals' preference toward fluid—for example, mice avoid drinking lemon juice. Interestingly, when the team stimulated sour cells with light, they did not observe that kind of aversive behavior in the engineered mice.

"These results raise the question: What information about taste are sour cells really relaying to the brain?" Zocchi says. "Maybe sour cells are not directly linked to the unpleasant sourness that we perceive, but instead they may induce a different type of taste, like water, when stimulated."

"It's important to note that stimulation of these cells does not alleviate thirst," says Oka. "But this finding helps us understand how the brain interprets water signals under normal and thirsty states. Next, we would like to tackle the mechanisms by which the hedonic value or 'pleasantness' of sensory inputs are regulated by brain activity."

The paper is titled "The cellular mechanism for water detection in the mammalian taste system." Gunther Wennemuth of Duisburg-Essen University is a co-author. Funding was provided by startup funds from the president and provost of Caltech and Caltech's Division of Biology and Biological Engineering, the Searle Scholars Program, the Edward Mallinckrodt, Jr. Foundation, the Okawa Foundation, the McKnight Foundation, and the Klingenstein-Simons Fellowship Award.

The Cost of "Living": How Viruses Hijack a Host's Energy Supply

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The energetic burden of a viral infection on the host
News Writer: 
Lori Dajose
Two viruses on a scale
A host will lose the same amount of energy to create two very different viruses: T4, which infects bacteria, and influenza, which infects mammals like humans. But compared to the host's energy budget, T4 takes up a much more substantial fraction of energy than influenza.
Credit: Caltech

Viruses occupy a strange no-man's-land between the living and the nonliving. In order to reproduce, they must infect a living host and hijack its resources. But while it is understood that this parasitic relationship can lead to disease and death, few quantitative studies have examined the energetic cost of viral infections relative to the host's energy economy.

A new study by Caltech researchers now provides the first quantitative estimates of the energetic burden of various viral processes across different types of viral infections. The work provides critical insights into the energy constraints guiding viral life cycles and evolution, which could ultimately lead to better vaccines and treatments.

The work was done in the laboratory of Rob Phillips, Fred and Nancy Morris Professor of Biophysics and Biology in both the Division of Biology and Biological Engineering and the Division of Engineering and Applied Science. It appears in a paper in the May 16 issue of the Proceedings of the National Academy of Sciences.

Many viruses, such as the ones examined in this paper, are essentially spherical shells called capsids containing genetic material. When a virus infects a host cell, it uses the cell's machinery to make copies of its own genetic material in a process called replication. During the stage known as translation, this genetic material is used to produce the proteins that will form the capsid. Finally, during the process of self-assembly, that protein shell and the copies of the virus's genetic material are assembled into new viruses. At some point, after many such viruses have been synthesized within the host, the host cell reaches its burst size—the size at which newly created viruses burst out of the cell and go on to infect new hosts. In principle, infection spreads faster as the burst size becomes larger.

Phillips, along with first author and graduate student Gita Mahmoudabadi and their colleagues, set out to quantify how much energy is stolen from the host during each of these processes—and how that may set a limit on the number of viruses produced and released by an infected cell.

The team looked at two different kinds of viruses: T4, a so-called bacteriophage virus, which uses DNA as its genetic material and infects bacteria; and the influenza virus, which uses RNA to carry its genetic information and infects mammalian cells. Using their estimates for the energy it takes to build one virus and for the burst sizes of the two viruses, they calculated the total cost to the host of the infection.

The researchers found that the bacteriophage usurped roughly 30 percent of its host organism's energetic resources and produced about 200 viruses before bursting out of the cell. The influenza virus, on the other hand, siphoned away only about 1 percent of its host cell's energy but created about 6,000 new viruses.

"You might think that a virus would want to drain as much energy from the host as possible in order to maximize its burst size," Mahmoudabadi says. "But our estimates show that this strategy is most befitting viruses that infect single-celled organisms such as bacteria. We were surprised to learn that viruses infecting multicellular organisms, such as those responsible for the seasonal flu, take up a small fraction of the host energy supply. We think this might be reflective of influenza's strategy to ensure both its immediate and long-term survival by sparing its multicellular host."

These studies provide a foundation for further work aimed at disentangling the complicated set of interactions that govern the winners and losers in this ever-present warfare between organisms and their pathogens.

The paper is titled "The energetic cost of building a virus." Ron Milo of the Weizmann Institute of Science is a co-author. Funding was provided by the National Science Foundation, the John Templeton Foundation, and the National Institutes of Health.

Overriding the Urge to Sleep

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New research identifies a group of neurons that can help us stay awake when it matters.
News Writer: 
Lori Dajose
An image of neurons chemically altered to glow red, blue, and green.
Dopaminergic neurons genetically engineered to express fluorescing genes. The fluorescence is used to measure activity of the neurons, which have been implicated in the sleep/wake cycle.
Credit: Courtesy of the Gradinaru laboratory

Caltech researchers have identified a neural circuit in the brain that controls wakefulness. The findings have implications for treating insomnia, oversleeping, and sleep disturbances that accompany other neuropsychiatric disorders, such as depression.

The work was done in the laboratory of Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering, Heritage Medical Research Institute Investigator, and director of the Center for Molecular and Cellular Neuroscience of the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. It appears in the June 8 online edition of the journal Neuron.

Gradinaru and her team wanted to know: How do we overcome tiredness in the face of a looming deadline or rouse ourselves in the dead of night to feed a crying baby? In other words, in the face of so-called salient stimuli, how do we override the natural drive to sleep?

"To answer this question, we decided to examine a region of the brain, called the dorsal raphe nucleus, where there are an under-studied group of dopamine neurons called dorsal raphe nucleus neurons, or DRNDA neurons," says Gradinaru. "People who have damage in this part of their brain have been shown to experience excessive daytime sleepiness, but there was not a good understanding of the exact role of these neurons in the sleep/wake cycle and whether they react to internal or external stimuli to influence arousal."

The team studied DRNDA neurons in mice, which are a model organism for studying the human brain. First, the team measured DRNDA activity while the animals encountered salient stimuli, such as the arrival of a potential mating partner, or a sudden unpleasant sensation, or food. The DRNDA neurons were highly active during these events, which led the researchers to theorize that the neurons send signals of salience and arousal, which can then modulate the state of sleep or wakefulness.

"We then measured DRNDA activity throughout the sleep/wake cycle and found that these neurons are least active when the animal is sleeping and increase in activity as the animal is waking up," says Ryan Cho, a graduate student and the first author on the paper. "We aimed to discover whether this was just a correlation or if the activity of the neurons was actually causing changes in sleep-wake states."

The researchers used a technique called optogenetics to engineer DRNDA cells to be stimulated by light. After stimulating these neurons with light during the time that the animal would normally sleep, Gradinaru and her team found that the mouse woke up from sleep and remained awake. The reverse was true when the activity of DRNDA was chemically silenced—the animal was likely to fall asleep, even in the face of motivationally important stimuli, such as the odor of a predator or a mating partner. This implied that activity of the DRNDA neurons truly governed sleep-wake behaviors.

Finally, the researchers examined the role of these neurons in awaking due to external stimuli. The neurons' activity was silenced with optogenetics, and a loud noise was played while the animals were asleep. Whereas control mice often woke up, the mice with blocked DRNDA often ignored the sound and remained asleep.

"These experiments showed us that DRNDA cells are necessary for full wakefulness in the face of important stimuli in mice," Gradinaru says. "DRNDA neurons are found analogously in humans, and while they have not been studied in depth, their degeneration has been correlated with excessive daytime sleepiness in patients with neurodegenerative disorders such as multiple systems atrophy and Lewy body dementia. Further work is necessary to establish causation in humans and to test the potential of the DRNDA as a therapeutic target for insomnia or oversleeping, and for sleep disturbances that accompany other psychiatric disorders such as depression, bipolar disorder, and schizophrenia."

The paper is titled "Dorsal Raphe Dopamine Neurons Modulate Arousal and Promote Wakefulness by Salient Stimuli." Other co-authors are postdoctoral scholars Jennifer Treweek (BS '04), Alon Greenbaum, and J. Elliott Robinson; former senior research scientist Cheng Xiao; and Caltech lecturer Lindsay Bremner. Funding was provided by the National Institutes of Health (NIH), the NIH's National Institute on Aging, the Heritage Medical Research Institute, the Pew Charitable Trusts, the Michael J. Fox Foundation, the Caltech-Gwangju Institute of Science and Technology exchange program, and the Alfred P. Sloan Foundation.


A New Approach to Biology

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Professor Rob Phillips reinvents the freshman biology course.
News Writer: 
Lori Dajose
Rob Phillips giving a Bi 1 lecture.
Rob Phillips giving a Bi 1 lecture.
Credit: Caltech

Why haven't insects multiplied enough to eat all the leaves on every tree—that is, why do trees stay green? How are atoms taken from inorganic materials to make living matter? What is the fastest rate at which a cell can divide?

These are some of the "puzzles" posed to undergraduate students in the introductory course Bi 1: Principles of Biology, taught this year by Rob Phillips, the Fred and Nancy Morris Professor of Biophysics and Biology in the divisions of Engineering and Applied Science and Biology and Biological Engineering.

As part of the Caltech undergraduate core curriculum, every student must fulfill a biology requirement. Many do so by taking Bi 1. The challenge facing Phillips was, in his own words: "What is the best way to teach biology to 220 non-biology majors?"

"I believe the most fascinating subject of our time is the quantitative study of living matter, trying to understand the living part of the world with the same precision as we have understood the inorganic world," he says. "Many students think of biology as a subject that is all about a variety of facts. I reject this viewpoint and in teaching this class I aimed to find an alternative to the 'death by powerpoint' approach."

Instead of a traditional encyclopedic approach where students learn about the broad sweep of facts that have been accumulated about the living world, Phillips decided to focus the course on what he calls the "broad guiding principles of biology," including lectures on biogeography (the study of the geographic distribution of species), the role of predators' ecosystems, evolution, the fossil record, photosynthesis, gene regulation, and how probability informs our understanding of biological mechanisms.

"I wanted to cultivate a sense of wonder," he says. "I want students to be observers of nature, and not necessarily adhere to manmade, artificial divisions of biological topics." Thus, each lecture began with a so-called puzzle. These were not rhetorical questions—they could be solved with techniques from biology, computer science, mathematics, chemistry, and physics. Homework questions were similar, requiring order-of-magnitude calculations made using both data and intuition to solve problems such as "how many sea otters does a killer whale need to eat every day?" to illustrate how keystone species—predators that hold ecosystems together—work.

Students were also encouraged to go outside to observe and reflect on the natural world. While this was not a formal assignment, Phillips suggested that the students think of sentences beginning with "I wonder…" and try to figure out what physical constraints set the scale of different objects in the natural world—such as, what sets the scale of how tall a tree can be, how long it took whales to evolve, or how far a bird can fly without eating.

"I found that doing the actual computations gave me a much more solid understanding of biology than just memorizing facts," says Sophia Coplin, a freshman computer science major. "We were using programming and statistical mechanics and other science to calculate the size of a bacterium, how evolution works, how a cell uses energy on a molecular level—all kinds of things. We were really thinking hard about and solving biological problems."

"I believe that biology is a forum for all scientists," says Phillips, who received an ASCIT (Associated Students of the California Institute of Technology) Teaching Award for his work. "Especially here at Caltech, where we don't steer clear of math and chemistry and physics to solve problems. Many of the greatest problems facing humanity, both in terms of curiosity and in terms of impact on our civilization, have to do with understanding the workings of the living world. Bi 1 as a course has the capacity to bring a huge set of brilliant students into thinking about subjects that they may have thought were not of interest to them."

Lights Out: The Neural Relationship Between Light and Sleep

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How light directly affects sleeping and waking
News Writer: 
Lori Dajose
Galanin expression in the brain
Overexpression of the neuropeptide prokineticin 2 (Prok2) results in an increase in expression of the sleep-promoting neuropeptide galanin (shown in white) when zebrafish are kept in the light, but not when they are kept in the dark, compared to wild type control animals. These images show galanin expression in the anterior hypothalamus, a brain region known to promote sleep in mammals.
Credit: Courtesy of the Prober laboratory

Humans are diurnal animals, meaning that we usually sleep at night and are awake during the day, due at least in part to light or the lack thereof. Light is known to affect sleep indirectly by entraining—modifying the length of—our circadian rhythms and also rapidly and directly due to a phenomenon known as masking. But while a great deal is known about how light affects circadian rhythms, little is known about the direct effects of light on sleep: Why do we tend to wake up if the lights are flipped on in the middle of the night? Why does darkness make us sleepy? Caltech researchers in the laboratory of Professor of Biology David Prober say they have discovered at least part of the answer: a specific protein in the brain that responds to light and darkness to set the correct balance between sleep and wakefulness.

Their work is described in a paper appearing online in the journal Neuron on June 22.

"Researchers had previously identified the photoreceptors in the eye that are required for the direct effect of light on wakefulness and sleep," says Prober. "But we wanted to know how the brain uses this visual information to affect sleep."

The Prober laboratory uses zebrafish as a model organism for studying sleep. The animals are optically transparent, allowing for noninvasive imaging of their neurons; they also have a diurnal sleep/wake pattern like that of humans. To investigate how their sleep responds to light, Wendy Chen, a former graduate student in Prober's lab, led studies examining a particular protein in the zebrafish brain called prokineticin 2 (Prok2).

Chen genetically engineered zebrafish to overexpress Prok2, resulting in an abundance of the protein. She found that in contrast to normal zebrafish, these animals were more likely to fall asleep during the day and to wake up at night. Surprisingly, the effects did not depend on the engineered fish's normal circadian sleep/wake cycle but rather depended only on whether the lights were on or off in their environment. These observations suggest that an excess of Prok2 suppresses both the usual awakening effect of light and the sedating effect of darkness.

Chen then generated zebrafish with mutated forms of Prok2 and its receptor, and observed light-dependent sleep defects in these animals. For example, Chen found that zebrafish with a mutated Prok2 receptor were more active when the lights were on and less active when the lights were off, the opposite of what she had observed in animals that overexpressed Prok2 and had functional Prok2 receptors.

"Though diurnal animals such as zebrafish spend most of their time asleep at night and awake during the day, they also take naps during the day and occasionally wake up at night, similar to many humans," Prober says. "Our study's results suggest that levels of Prok2 play a critical role in setting the correct balance between sleep and wakefulness during both the day and the night."

Next, the researchers wanted to know how Prok2 was modulating light's effects on sleep. To answer this question, they decided to examine whether other proteins in the brain that are known to affect sleep were required for the effects of Prok2 on sleep behavior. They found that the sedating effect of Prok2 overexpression in the presence of light requires galanin, a known sleep-promoting protein. They also found that Prok2 overexpression increased the level of galanin expression in the anterior hypothalamus, a key sleep-promoting center in the brain. But in animals that were engineered to lack galanin, overexpression of Prok2 did not increase sleep.

These findings provide the first insights into how light may interact with the brain to affect sleep and provide a basis for scientists to begin exploring the genes and neurons that underlie the phenomenon. However, further work is needed to fully explain how light and dark directly affect sleeping and waking, and to determine whether Prok2 has a similar function in humans. If it does, this work might eventually lead to new sleep- and wake-promoting drugs.

The paper is titled "Light-dependent regulation of sleep/wake states by prokineticin 2 in zebrafish." Other Caltech co-authors are postdoctoral scholars Chanpreet Singh and Grigorios Oikonomou. Sabine Reichert and Jason Rihel of University College London also contributed to the study. The work was funded by the National Institutes of Health; the Edward Mallinckrodt, Jr. Foundation; the Rita Allen Foundation; and the Brain & Behavior Research Foundation.

Novel Viral Vectors Deliver Useful Cargo to Neurons Throughout the Brain and Body

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The viruses reach their targets via the bloodstream and could be used to treat neural disorders
News Writer: 
Lori Dajose

Delivering Genes Across the Blood-Brain Barrier

Delivering Genes Across the Blood-Brain Barrier
Delivering Genes Across the Blood-Brain Barrier
Credit: Chan et. al., Gradinaru Lab / Caltech

Viruses have evolved to be highly effective vehicles for delivering genes into cells. Seeking to take advantage of these traits, scientists can reprogram viruses to function as vectors, capable of carrying their genetic cargo of choice into the nuclei of cells in the body. Such vectors have become critical tools for delivering genes to treat disease or to label neurons and their connective fibers with fluorescent colors to map out their locations. Because viral vectors have been stripped of their own genes and, thereby, of their ability to replicate, they are no longer infectious. Therefore, achieving widespread gene delivery with the vectors is challenging. This is especially true for gene delivery to hard to reach organs like the brain, where viral vectors have to make their way past the so-called blood-brain barrier, or to the peripheral nervous system, where neurons are dispersed across the body.

Now, to enable widespread gene delivery throughout the central and peripheral nervous systems, Caltech researchers have developed two new variants of a vector based on an adeno-associated virus (AAV): one that can efficiently ferry genetic cargo past the blood-brain barrier; and another that is efficiently picked up by peripheral neurons residing outside the brain and spinal cord, such as those that sense pain and regulate heart rate, respiration, and digestion. Both vectors are able to reach their targets following a simple injection into the bloodstream. The vectors are customizable and could potentially be used as part of a gene therapy to treat neurodegenerative disorders that affect the entire central nervous system, such as Huntington's disease, or to help map or modulate neuronal circuits and understand how they change during disease.

The work was done in the laboratory of Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering, Heritage Medical Research Institute Investigator, director of the Center for Molecular and Cellular Neuroscience in the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech, and principal investigator of the Beckman Institute's CLOVER (CLARITY, Optogenetics, and Vector Engineering Research) Center.

A paper describing the research appears online in the June 26 issue of Nature Neuroscience.

"We have now developed a new collection of viruses and tools to study the central and peripheral nervous systems," says Gradinaru. "We are now able to get highly efficient brain-wide delivery with just a low-dose systemic injection, access neurons in difficult-to-reach regions, and precisely label cells with multiple fluorescent colors to study their shapes and connections."

Gradinaru and her team modified the external surface of an AAV developed in 2016, engineering the virus's shell, or capsid, to allow it to more efficiently deliver genes to cells in the brain and spinal cord following intravenous injection. They named the new virus AAV-PHP.eB.

The team also developed an additional capsid variant they call AAV-PHP.S, which is able to transduce peripheral neurons.

"Neurons outside of the central nervous system have many functions, from relaying sensory information to controlling organ function, but some of these peripheral neural circuits are not yet well understood," says Ben Deverman, senior research scientist and director of the Beckman Institute's CLOVER Center. "The AAV-PHP.S vector that we developed could help researchers study the activity and function of specific types of neurons within peripheral circuits using genetically-encoded sensors and tools to modulate neuronal firing with light or designer drugs, respectively."

The new AAV vectors can also deliver genes that code for colorful fluorescent proteins; such proteins are useful in identifying and labeling cells. In this process, multiple AAVs—each carrying a distinct color—are mixed together and injected into the bloodstream. When they reach their target neurons, each neuron receives a unique combination of colors, thereby giving it a visually distinct hue that makes it easier for the researchers to distinguish its fine details from those of its neighbors. Furthermore, the team devised a technique to control the number of neurons labeled—labeling too many neurons makes it impossible to distinguish individual ones—that allows researchers to visualize individual neuron shapes and trace their connecting fibers through intact tissues using another technology the Gradinaru laboratory has helped develop, known as tissue clearing.

"Usually, when researchers want a mouse or other animal model to express fluorescent proteins in certain cells, they need to develop genetically modified animals that can take months to years to make and characterize," says former graduate student and first author Ken Chan (PhD '17). "Now with a single injection, we can label specific cells with a variety of colors within weeks after the injection."

"For our new systemic viral vectors—AAV PHP.S and AAV PHP.eB—there are many potential uses, from mapping circuits in the periphery and fast screening of gene regulatory elements to genome editing with powerful tools such as CRISPR-Cas9," says Gradinaru. "But perhaps the most exciting implication is that our tools, when paired with appropriate activity modulator genes, could enable non-invasive deep brain modulation for the treatment of neurological diseases such as Parkinson's disease."

The paper is titled "Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems." In addition to Chan, Deverman, and Gradinaru, other coauthors are postdoctoral scholars Min Jang, Alon Greenbaum, Luis Sánchez-Guardado, and Wei-Li Wu; graduate student Bryan Yoo; undergraduate student Namita Ravi; Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology and a Heritage Medical Research Institute Investigator; and research professor Carlos Lois. Funding was provided by the National Institutes of Health, the Presidential Early Career Award for Scientists and Engineers, the Heritage Medical Research Institute, the Beckman Institute and Rosen Center at Caltech, the Gordon and Betty Moore Foundation, the Shurl & Kay Curci Foundation, the Hereditary Disease Foundation, the Friedreich's Ataxia Research Alliance (FARA) and FARA Australasia, and the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office.

Speech and Transgenic Songbirds

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New genomic tools will enable the study of higher cognitive functions in songbirds
News Writer: 
Lori Dajose
Carlos Lois
Carlos Lois
Credit: Caltech

Research Professor Carlos Lois is one of 10 recipients of an EDGE (Enabling Discovery through Genomic Tools) grant from the National Science Foundation (NSF). The newly formed NSF EDGE program will enable biologists to develop enhanced genomic tools that could one day reveal insights into why organisms are structured the way they are and function the way they do.

The Lois laboratory is particularly interested in developing new methods of producing transgenic zebra finches, genetically modified songbirds that are commonly studied to provide understanding of vocal learning and communication. The anatomical organization of these birds' brains shows similarities to the organization of vocal control centers in humans, and the manner by which they learn song is similar to how humans learn to speak.

"Unfortunately, many of the most interesting questions related to higher cognitive functions cannot be studied in any of the model organisms—flies, mice, fish, and worms—because their behavioral repertoires are quite limited," says Lois. "For example, none of these model organisms are useful for investigating the genetic basis of language and speech. Now, for the first time, transgenic songbirds are allowing us and others to generate animal models of human diseases affecting higher cognitive functions and communication disorders, such as autism, schizophrenia, and speech disorders."

The work will be a collaboration between Caltech, The Rockefeller University, and Oregon Health & Science University.

The Allen Discovery Center for Cell Lineage Tracing

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A new collaboration between Caltech, the University of Washington in Seattle, and Harvard University aims to develop new in-cell recording technologies to produce genomic maps of multi-cellular development
News Writer: 
Lori Dajose
MEMOIR
MEMOIR enables the histories of cells to be recorded in their genomes and then read out using microscopy. Here, MEMOIR cells were variably activated, as seen by the bright cyan nuclear fluorescence in some cells. The cells recorded information in response to this signal with the help of a DNA-editing system called CRISPR. This recorded information was then read out using a technique called seqFISH to visualize certain RNA transcripts in the cells (red dots).
Credit: Elowitz and Cai Labs/Caltech

Three researchers from Caltech—Michael Elowitz, professor of biology and bioengineering, Howard Hughes Medical Institute Investigator, and executive officer for biological engineering; Long Cai, research professor; and Carlos Lois, research professor—have received funding to create the Allen Discovery Center for Cell Lineage Tracing in collaboration with the University of Washington in Seattle and Harvard University. Support for the establishment of the center comes from the Paul G. Allen Frontiers Group, which will provide $10 million over four years with the potential for $30 million over eight years.

The goal of the Allen Center will be to use newly developed technologies to create global maps of cellular development, tracing cells as they divide, move, and differentiate throughout an organism's development, and revealing the relationships between the vast number of diverse cells that make up a single organism.

Last year, Elowitz and Cai collaborated on a gene-editing technique called MEMOIR, which allows cells to record information in their own genomes, in a format that can be read out by microscopic imaging.                                                                                                                     

"The goal is for each cell to be able to tell us its own individual history, including its lineage and the specific molecular events it has experienced during development," says Elowitz. "To do this, we decided to team up with groups from the University of Washington in Seattle and Harvard University who were developing related technologies, and work together to map lineage and developmental histories of cells."

"One of the fundamental questions in biology is to understand how a single-celled embryo gives rise to a complex animal with hundreds of different cell types organized into highly complex organs and tissues," says Lois, who will be one of the center's principal investigators. "A critical step to solving this problem is to be able to track the progeny of cells—that is, their lineage. The funding from the Allen Discovery Center will allow us to develop new methods with which to reconstruct lineage trees over many generations. In addition, we will develop new strategies so that cells can write in their genomes a record of the molecular signals that control their fate. With these methods, the cells will, in a sense, keep a 'journal' of what is happening to them, and we will be able to read that journal in the genomes of their progeny many generations later. With this kind of information, we will be able to understand the molecular signals that determine, for example, that a single stem cell will give rise to two daughter cells, with one becoming a neuron that processes visual information while the other processes auditory information."

"Michael and I had this crazy idea over coffee to use synthetic biology to engineer a circuit to record the 'memoir' of a cell," says Cai, who will also be a principal investigator of the center. "My lab had just gotten a proof-of-principle experiment working to sequentially read out mRNA levels in the cell. So, we thought it would be good to apply that method to read out the recorded 'bits' in the cell. That was 2013 and the project has worked beyond our initial conception of the idea. We owe all this to the creativity and the hard work of the people in our labs. The Discovery Center will allow us to answer fundamental questions about cell fate decision-making, which like real life involves many dimensions and is context dependent, by reading out the MEMOIR of those cells."

The Neural Codes for Body Movements

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A small patch of neurons fires in complex ways to encode movement of much of the body
News Writer: 
Lori Dajose
A screen with signals
The study participant watches the activity of one of her neurons in real time. Brain-machine interfaces can interpret the neural activity of a subject's thoughts and translate them into robotic movement.
Credit: Caltech

A small patch of neurons in the brain can encode the movements of many body parts, according to researchers in the laboratory of Caltech's Richard Andersen, James G. Boswell Professor of Neuroscience, Tianqiao and Chrissy Chen Brain-Machine Interface Center Leadership Chair, and Director of the T&C Brain-Machine Interface Center of the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. Understanding this neural code could help improve the lives of people with paralysis or with motor deficits from neurological diseases such as a stroke.

The findings appear in a paper available online in the July 20 edition of Neuron.

The motor cortex, the region of the brain that governs movement, lies at the end of the sensory-to-motor pathway and controls the muscles of the body. Earlier on that pathway is the posterior parietal cortex (PPC), a high-level cognitive area that encodes the intention to move. When a person intends to drink from a glass of water, for example, the signal for this intention in the PPC is transmitted to the motor cortex, which sends signals down the spinal cord and to the proper limbs. In 2015, Andersen and collaborators successfully implanted tiny prosthetic devices into the PPC of paralyzed patients; these so-called neuroprosthetics measure the movement intentions of the patient—to pick up a cup to take a drink, for example—and execute those movements accurately with a robotic arm.

For the current work, the researchers aimed to discover how the PPC encodes and organizes neural information about other body movements, such as grasping a cup with the left versus right hand, or imagining versus attempting certain bodily movements.

To do this, the researchers implanted a four-by-four-millimeter chip composed of 96 electrodes into a subdivision of the PPC called the anterior intraparietal area (AIP), to measure the neural activity of a tetraplegic human who volunteered to take part in a brain-machine interface clinical trial. Traditionally, the AIP has been thought to specialize in grasping objects. However, the researchers found that AIP coded for more than just grasps.

"We found that different neurons in the AIP were indeed selective for different grasps, but we also found activation for shoulder or hand movements, whether imagined or attempted, and for either side of the body," Andersen says. "In fact, a portion of the cells were even tuned to speech movements. This was truly amazing to find so much information contained in such a small population of neurons."

"This compact code was made possible by so-called mixed encoding," says co-lead author Tyson Aflalo, senior scientific researcher at Caltech and executive director of the T&C Brain-Machine Interface Center. "In mixed coding, single neurons will respond to a diverse mixture of actions, or variables. For instance, a single neuron may respond for imagined movement of both the left hand and right shoulder. One breakthrough of our study is that we found that mixed-codes can be highly structured."

Previous descriptions of mixed coding assumed that variables were randomly mixed among neurons. However, Andersen's team discovered that there is a highly structured organization of the mixing of variables that is determined by the part of the body, or effector, being moved.

"The effectors are encoded in a largely independent manner within the neural population—a property we call functional segregation," says Caltech graduate student and co-lead author Carey Zhang. "Body side and cognitive strategy—whether the subject attempted or only imagined movement—are organized (highly correlated) within the effector representations. For instance, if a neuron responds to a right hand attempted movement activation, it is also more likely to respond to a left hand attempted movement."

"The functional segregation of effectors may provide for more efficient neural computations and learning," says Andersen. "For instance, because hand dynamics are similar for the left and right hand, learning with one hand should easily be transferred to the other."

Thanks to the functional segregation, Zhang says, learning a new hand gesture only affects the hand and would not produce spurious movements of the shoulder, for example.

According to the researchers, this area of the PPC is likely still primarily oriented toward grasp processing. However, the PPC is connected to many other areas—it is a so-called association cortical area—and thus the multitude of body signals may reflect the coordination of multiple cortical areas. This provides an advantage from a neural prosthetics perspective, Andersen says, as a small implant sampling a small number of neurons can provide information for many types of intended movements. 

The overlap of the cognitive strategies of imagined and attempted movement has important implications. "Athletes such as gymnasts often imagine sequences of movements and use that for training," Aflalo says. "We have found that this imagination is actually activating the same neural circuits that govern the movement itself—though not in an entirely identical way, so we can still tell them apart."

"The hope is that this understanding of the neural code in the PPC can be applied not only to paralysis but to other kinds of motor deficits from neurological diseases, such as difficulty moving after a stroke," says Andersen. "That's our main thrust, to improve the lives of people with traumatic brain injury, strokes, peripheral neuropathies, and other kinds of paralytic diseases."

The paper is titled "Partially Mixed Selectivity in Human Posterior Parietal Association Cortex." Another Caltech coauthor is former graduate student Boris Revechkis (PhD '15). Funding was provided by the National Institutes of Health, the Tianqiao and Chrissy Chen Brain-Machine Interface Center at Caltech, the Della Martin Foundation, the Caltech Conte Center for Social Decision Making, and the James G. Boswell Foundation.

Fighting Viruses with Viruses

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How genetically engineered viruses develop into effective vaccines
News Writer: 
Lori Dajose
HIV (yellow) infecting a human cell
HIV, the AIDS virus (yellow), infecting a human cell
Credit: Public Domain

Lentiviral vectors are virus particles that can be used as a vaccine to stimulate the immune system to fight against specific pathogens. The vectors are derived from HIV, rendered non-pathogenic, and then engineered to carry genetic material into the body's immune cells; the genes program the cells to fight specific pathogens. New research from the laboratory of David Baltimore, president emeritus and the Robert Andrews Millikan Professor of Biology, shows that additional materials acquired during the vector's formation—namely, proteins and human genomic DNA—play an important role in activating the immune system. Understanding how lentiviral vectors interact with the immune system is critical to improving vaccines so as to maximize the immune response, and to understanding infectious processes occurring in natural pathogens.

The work is described in a paper appearing in the online version of Science Immunology on July 21.

Lentiviral vectors can be engineered to introduce the genetic material of an antigen—a molecule that triggers an immune response—specifically into a type of immune cell known as a dendritic cell. Dendritic cells present these antigens to T cells and B cells, which then develop an antigen-specific immune memory—meaning they will recognize and respond if they encounter the antigen in the future.

Scientists create lentiviral vectors using specially engineered human cell lines. As the vector particles form within those genetically programmed cells, the vectors encapsulate not only the genetic material of the antigen of interest but other material from the host cell, such as the antigenic proteins themselves and genomic DNA from the human cells.

"The primary way of delivering the antigen into dendritic cells is by delivering its genetic material so that the cell can produce the antigenic protein itself," says first author Jocelyn Kim (PhD '16), a Caltech graduate student at the time the work was done and now an assistant professor at UCLA. "We wanted to understand whether the delivery of the antigen protein itself and genomic DNA contained in vector preparations was affecting the immune response."

The team discovered that, indeed, delivering the antigenic protein itself into a target cell along with non-viral genetic material enhances the immune response. Additionally, they found that the human genomic DNA that is inadvertently encapsulated into the vector is important in activating the immune response.

"It is likely that the presence of human nuclear DNA in the cytoplasm of the dendritic cell triggers an immune response, because the cell recognizes that nuclear DNA should not be in the cytoplasm," Kim says.

"We started this work to try to understand why this form of immunization works so well," says Baltimore. "We found many surprises, showing that it is a much more complex process than we imagined. We are now pursuing work to use this form of immunization with the hope that our knowledge of the complexity will improve the effectiveness of the immunization process as a form of cancer therapy."

The paper is titled "Dendritic cell-targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation." Other Caltech coauthors include Rajan Kulkarni, Visitor in Biology & Biological Engineering; Kevin Lee, former Baltimore lab technician; former graduate student Geoffrey Lovely (PhD '14), former research assistant Yong Ouyang, and former graduate student Lili Yang (PhD '04). Funding was provided by the National Institutes of Health and the American Foundation for AIDS Research.


Caltech, Cornell Create Creature-Cataloging Contest for Computers

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Competition promoted development of more powerful machine- learning algorithms for visual identification of species
News Writer: 
Emily Velasco
A collage showing a variety of lifeforms
Algorithms developed for a contest held by Caltech and Cornell Tech can identify species that appear in photos.
Credit: Credit: Clockwise from top left-Jana Ohrner, Thomas Barbin, Clayton Orsay, Caltech, Steve Harbula, and John Sullivan. (CC BY-NC)

At a glance, would you be able to tell the difference between a donkey and a mule? A jaguar and a leopard? Most computers can't, at least not yet, but a contest hosted by Caltech and Cornell Tech, the engineering campus of Cornell University, aimed to change that.

The two institutions teamed up to create the iNaturalist Challenge, a competition to create the best machine-learning algorithm for identifying the world's plant and animal species. The contest was an outgrowth of the institutions' previous work together on Visipedia, a visual encyclopedia created by a network of people and machine-learning computers that harvest image information off the internet. The technology was developed for the encyclopedia by Pietro Perona's Vision Group in the Division of Engineering and Applied Science at Caltech and Serge Belongie's Computer Vision Group at Cornell Tech. It has previously been used in Registree, a piece of software that identifies and catalogs urban tree species, and Merlin Bird ID, a mobile app that can identify 650 species of North American birds from photos taken by the user.

Grant Van Horn and Oisin Mac Aodha, researchers in Perona's lab, wanted to see how much further they could push machine-learning technology, and the idea for the contest was born. During a period stretching from April to July, anyone who wanted to compete was given access to a database of 650,000 images of more than 5,000 species in categories that included, among others, protozoans, fungi, plants, archanids, and mammals. They used that database to develop algorithms for automatic species identification.

By the time the competition closed in July, the organizers had received 32 entries comprising teams and individuals. Van Horn says the winning algorithm was able to correctly identify species in a test database of 100,000 photos 80 percent of the time when it was allowed only one guess per photo. When the algorithm was allowed five guesses per photo, its success rate rose to 95 percent.

"That's way better than almost every person would do on a test like this," he says. "It's 5,000 species, so it's pretty hard to be an expert on all of them. This is where we're seeing the true benefit of computer vision."

Still, Mac Aodha says, while impressive, the algorithms developed for the contest still use traditional neural network technology and are far from perfect. He hopes future contests will spur something new in the machine-learning field.

"From the competition results we can see that computers perform very well when they have access to many photographs of a given species. However, to truly reach expert human ability, we need to design algorithms that are capable of understanding new species from only a small number of images," he says.

"Building on our success in identifying birds with Visipedia, we challenged technologists to develop algorithms to recognize even more species with incredible results," said Serge Belongie, professor of computer science at Cornell Tech and the co-founder of Visipedia. "It's another step forward in computer vision technologies."

Support for the contest came from iNaturalist, a website that allows users to submit photos of plants and animals to be identified by a community of citizen scientists. iNaturalist provided the image databases, while funding was provided by Google. Prizes included Google Cloud credits and a graphics processing unit supplied by NVIDIA. More information about the competition and the results can be viewed here.

Medicine of the Future: New Microchip Technology Could Be Used to Track Smart Pills

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Caltech researchers have developed microscale devices that relay their location in the body
News Writer: 
Whitney Clavin
illustration of chip in gut
Illustration of an ATOMS microchip localized within the gastrointestinal tract. The chip, which works on principles similar to those used in MRI machines, is embodied with the properties of nuclear spin.
Credit: Ella Marushchenko for Caltech

Researchers at Caltech have developed a prototype miniature medical device that could ultimately be used in "smart pills" to diagnose and treat diseases. A key to the new technology—and what makes it unique among other microscale medical devices—is that its location can be precisely identified within the body, something that proved challenging before.

"The dream is that we will have microscale devices that are roaming our bodies and either diagnosing problems or fixing things," says Azita Emami, the Andrew and Peggy Cherng Professor of Electrical Engineering and Medical Engineering and Heritage Medical Research Institute Investigator, who co-led the research along with Assistant Professor of Chemical Engineering and Heritage Medical Research Institute Investigator Mikhail Shapiro. "Before now, one of the challenges was that it was hard to tell where they are in the body."

A paper describing the new device appears in the September issue of the journal Nature Biomedical Engineering. The lead author is Manuel Monge (MS '10, PhD '17), who was a doctoral student in Emami's lab and a Rosen Bioengineering Center Scholar at Caltech, and now works at a company called Neuralink. Audrey Lee-Gosselin, a research technician in Shapiro's lab, is also an author.

Called ATOMS, which is short for addressable transmitters operated as magnetic spins, the new silicon-chip devices borrow from the principles of magnetic resonance imaging (MRI), in which the location of atoms in a patient's body is determined using magnetic fields. The microdevices would also be located in the body using magnetic fields—but rather than relying on the body's atoms, the chips contain a set of integrated sensors, resonators, and wireless transmission technology that would allow them to mimic the magnetic resonance properties of atoms.

"A key principle of MRI is that a magnetic field gradient causes atoms at two different locations to resonate at two different frequencies, making it easy to tell where they are," says Shapiro. "We wanted to embody this elegant principle in a compact integrated circuit. The ATOMS devices also resonate at different frequencies depending on where they are in a magnetic field."

"We wanted to make this chip very small with low power consumption, and that comes with a lot of engineering challenges," says Emami. "We had to carefully balance the size of the device with how much power it consumes and how well its location can be pinpointed."

The researchers say the devices are still preliminary but could one day serve as miniature robotic wardens of our bodies, monitoring a patient's gastrointestinal tract, blood, or brain. The devices could measure factors that indicate the health of a patient—such as pH, temperature, pressure, sugar concentrations—and relay that information to doctors. Or, the devices could even be instructed to release drugs.

"You could have dozens of microscale devices traveling around the body taking measurements or intervening in disease. These devices can all be identical, but the ATOMS devices would allow you to know where they all are and talk to all of them at once," says Shapiro. He compares it to the 1966 sci-fi movie Fantastic Voyage, in which a submarine and its crew are shrunk to microscopic size and injected into the bloodstream of a patient to heal him from the inside—but, as Shapiro says, "instead of sending a single submarine, you could send a flotilla."

The idea for ATOMS came about at a dinner party. Shapiro and Emami were discussing their respective fields—Shapiro engineers cells for medical imaging techniques, such as MRI, and Emami creates microchips for medical sensing and performing actions in the body—when they got the idea of combining their interests into a new device. They knew that locating microdevices in the body was a long-standing challenge in the field and realized that combining Shapiro's knowledge in MRI technology with Emami's expertise in creating microdevices could potentially solve the problem. Monge was enlisted to help realize the idea in the form of a silicon chip.

"This chip is totally unique: there are no other chips that operate on these principles," says Monge. "Integrating all of the components together in a very small device while keeping the power low was a big task." Monge did this research as part of his PhD thesis, which was recently honored with the Charles Wilts Prize by Caltech's Department of Electrical Engineering.

The final prototype chip, which was tested and proven to work in mice, has a surface area of 1.4 square millimeters, 250 times smaller than a penny. It contains a magnetic field sensor, integrated antennas, a wireless powering device, and a circuit that adjusts its radio frequency signal based on the magnetic field strength to wirelessly relay the chip's location.

"In conventional MRI, all of these features are intrinsically found in atoms," says Monge. "We had to create an architecture that functionally mimics them for our chip."

Sorting Molecules with DNA Robots

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A DNA nanorobot is programmed to pick up and sort molecules into predefined regions.
News Writer: 
Lori Dajose
Artist's illustration of DNA robots
Conceptual illustration of two DNA robots collectively performing a cargo-sorting task on a DNA origami surface, transporting fluorescent molecules with different colors from initially unordered locations to separated destinations. Considerable artistic license has been taken.
Credit: Demin Liu

Imagine a robot that could help you tidy your home: roving about, sorting stray socks into the laundry and dirty dishes into the dishwasher. While such a practical helper may still be the stuff of science fiction, Caltech scientists have developed an autonomous molecular machine that can perform similar tasks—at the nanoscale. This "robot," made of a single strand of DNA, can autonomously "walk" around a surface, pick up certain molecules and drop them off in designated locations.

The work was done in the laboratory of Lulu Qian, assistant professor of bioengineering. It appears in a paper in the September 15 issue of Science.

Why Nanobots?

"Just like electromechanical robots are sent off to faraway places, like Mars, we would like to send molecular robots to minuscule places where humans can't go, such as the bloodstream," says Qian. "Our goal was to design and build a molecular robot that could perform a sophisticated nanomechanical task: cargo sorting."

How to Build a Molecular Robot

Led by former graduate student Anupama Thubagere (PhD '17), the researchers constructed three basic building blocks that could be used to assemble a DNA robot: a "leg" with two "feet" for walking, an "arm" and "hand" for picking up cargo, and a segment that can recognize a specific drop-off point and signal to the hand to release its cargo. Each of these components is made of just a few nucleotides within a single strand of DNA.

In principle, these modular building blocks could be assembled in many different ways to complete different tasks—a DNA robot with several hands and arms, for example, could be used to carry multiple molecules simultaneously.

In the work described in the Science paper, the Qian group built a robot that could explore a molecular surface, pick up two different molecules—a fluorescent yellow dye and a fluorescent pink dye—and then distribute them to two distinct regions on the surface. Using fluorescent molecules enabled the researchers to see if the molecules ended up in their intended locations. The robot successfully sorted six scattered molecules, three pink and three yellow, into their correct places in 24 hours. Adding more robots to the surface shortened the time it took to complete the task.

"Though we demonstrated a robot for this specific task, the same system design can be generalized to work with dozens of types of cargos at any arbitrary initial location on the surface," says Thubagere. "One could also have multiple robots performing diverse sorting tasks in parallel."

Design through DNA

The key to designing DNA machines is the fact that DNA has unique chemical and physical properties that are known and programmable. A single strand of DNA is made up of four different molecules called nucleotides—abbreviated A, G, C, and T—and arranged in a string called a sequence. These nucleotides bond in specific pairs: A with T, and G with C. When a single strand encounters a so-called reverse complementary strand—for example, CGATT and AATCG—the two strands zip together in the classic double helix shape.

A single strand containing the right nucleotides can force two partially zipped strands to unzip from each other. How quickly each zipping and unzipping event happens and how much energy it consumes can be estimated for any given DNA sequence, allowing researchers to control how fast the robot moves and how much energy it uses to perform a task. Additionally, the length of a single strand or two zipped strands can be calculated. Thus, the leg and foot of a DNA robot can be designed for a desired step size—in this case, 6 nanometers, which is about a hundred millionth of a human's step size.

Using these chemical and physical principles, researchers can design not only robots but also "playgrounds," such as molecular pegboards, to test them on. In the current work, the DNA robot moves around on a 58-nanometer-by-58-nanometer pegboard on which the pegs are made of single strands of DNA complementary to the robot's leg and foot. The robot binds to a peg with its leg and one of its feet—the other foot floats freely. When random molecular fluctuations cause this free foot to encounter a nearby peg, it pulls the robot to the new peg and its other foot is freed. This process continues with the robot moving in a random direction at each step.

It may take a day for a robot to explore the entire board. Along the way, as the robot encounters cargo molecules tethered to pegs, it grabs them with its "hand" components and carries them around until it detects the signal of the drop-off point. The process is slow, but it allows for a very simple robot design that utilizes very little chemical energy.

Futuristic Applications

"We don't develop DNA robots for any specific applications. Our lab focuses on discovering the engineering principles that enable the development of general-purpose DNA robots," says Qian. "However, it is my hope that other researchers could use these principles for exciting applications, such as using a DNA robot for synthesizing a therapeutic chemical from its constituent parts in an artificial molecular factory, delivering a drug only when a specific signal is given in bloodstreams or cells, or sorting molecular components in trash for recycling."

The paper is titled "A cargo-sorting DNA robot." In addition to Thubagere and Qian, other co-authors are postdoctoral scholar Wei Li, graduate student Robert Johnson (BS '15), former visiting student Zibo Chen, former undergraduates Shayan Doroudi (BS '13), Yae Lim Lee (BS '12), Gregory Izatt (BS '14), and Sarah Wittman (BS '13); former graduate student Niranjan Srinivas (PhD '15), former senior research fellow Damien Woods; and Erik Winfree (PhD '98), professor of computer science, computation and neural systems, and bioengineering. Funding was provided by Caltech Summer Undergraduate Research Fellowships, the National Science Foundation, and the Burroughs Wellcome Fund.

Gut Bacteria May Play Role in Onset of Multiple Sclerosis

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The presence of “good-guy” and “bad-guy” microbes increases or decreases disease symptoms in mice
News Writer: 
Emily Velasco
An artist's rendering of gut bacteria.
An artist's rendering of gut bacteria.
Credit: Caltech

New research conducted at Caltech and UC San Francisco (UCSF) is suggesting that the key to curing multiple sclerosis (MS) could be hiding in an unlikely place—our poop. 

Multiple sclerosis is a disorder in which the body's own immune system induces an inflammatory response against the protective myelin sheath that helps neurons conduct signals. As the disease progresses, an afflicted individual will experience a range of symptoms that include muscle weakness, loss of sensation and coordination, digestive troubles, and depression. 

The cause of MS is not entirely clear, though genetics and viral infections have all been suspected of playing a role. The incidence of the disease is much higher in Western industrialized nations than elsewhere in the world, suggesting that other environmental factors, like time spent outdoors in the sun, have an effect as well.

It's also been theorized that the make-up of the gut microbiome—the collection of microbes that live in our digestive systems—could be involved, says Bryan Yoo, a Caltech grad student pursuing a degree in biology and bioengineering. The gut microbiome is a complex ecosystem of hundreds of distinct microorganisms, some beneficial and some potentially harmful. An out-of-balance gut biome with too few beneficial microorganisms or too many harmful ones has also been suspected of playing a role in cardiovascular disease, obesity, mental health, inflammatory bowel disease, and more.

"Maybe we're not getting enough of the microbial repertoire that our bodies need," Yoo says. "As far as MS goes, this is an intriguing frontier." 

In their study, which appears in the Proceedings of the National Academy of Sciences the week of September 11, Yoo and his co-authors, among them Caltech's Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology and Heritage Medical Research Institute Investigator, identify bacteria they believe have direct effects on the onset and severity of a mouse model that mimics the pathology of multiple sclerosis. 

At UCSF, researchers identified a group of MS patients who had not received any medical treatment for their disease and a group of healthy control individuals who lived with the MS patients (to control for environmental factors). They took fecal samples from each group and looked at the kinds of microbes found in each. Upon comparing those samples, the researchers found elevated levels of specific bacteria in MS patients. The control patients had elevated levels of a different species of bacteria. This suggested to the research team that bacteria prevalent in the MS patients might be having a pathogenic effect or that bacteria found in the control patients might be having a protective effect. 

At Caltech, researchers used mice engineered to be free of all microorganisms, including those that live in the gut. In one experiment, groups of the mice had an experimentally induced disease similar to MS. In the next experiment, they investigated the effects of specific microbes represented in MS and control patients on the immune system. 

In the first experiment, a group of mice were colonized with gut bacteria from the humans afflicted with MS and another group was inoculated with fecal samples from the healthy humans. When both groups were induced to have the MS-like disease, those that had their guts colonized with bacteria from MS patients experienced more severe disease outcomes. Furthermore, the mice that received the fecal sample from healthy control patients saw enhanced anti-inflammatory responses compared to mice that received samples from MS patients, potentially mitigating disease severity.  

In a second phase of the experiment, Caltech researchers worked with the bacteria identified by the UCSF team—Parabacteroides distasonis, which they suspected was easing MS symptoms, and Acinetobacter calcoaceticus and Akkermansia muciniphila, which they believed were making the disease worse.

Germ-free mice that did not have the MS-like disease were inoculated with those bacteria. One group received Parabacteroides and experienced reduced inflammatory responses. The groups of mice that received Acinetobacter and Akkermansia, in contrast, had increased inflammatory responses. 

The findings suggest that fecal transplants could hold promise as a treatment for patients with MS, Yoo says, cautioning that diseases like MS are multifaceted and likely the result of many factors besides the content of the gut.

"It's important that we look into the different causes of autoimmune diseases," he says. "Potentially there's a probiotic treatment out there for MS."

Yoo's paper, which was co-authored by Egle Cekanaviciute, a postdoctoral researcher at the UC San Francisco School of Medicine, is titled "Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models".

Other co-authors include Tessel F. Runia, Sneha Singh, Charlotte A. Nelson, Rachel Kanner, Stephen L. Hauser, Elizabeth Crabtree-Hartman, Bruce A. C. Cree and Sergio E. Baranzini of UCSF; Yun Kyung Lee and Sarkis K. Mazmanian of Caltech; Justine W. Debelius and Rob Knight of UC San Diego; and Ilana Katz Sand, Mar Gacias, Yungjiao Zhu and Patrizia Casaccia of the Icahn School of Medicine at Mount Sinai.

Funding for the research was provided by: the National Multiple Sclerosis Society, the NIH Institutional Research and Academic Career Development Award Postdoctoral Fellowship, the Department of Defense, the Valhalla Charitable Foundation, the Emerald Foundation, and the Heritage Medical Research Institute.

Interpreting Mixed (Molecular) Messages

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Understanding the language of cellular communication
News Writer: 
Lori Dajose
Artist depiction of ligands and receptors
In multicellular organisms, cells need to address messages to specific other cell types. To do so, cells use a set of similar, but not identical, signaling proteins, or ligands. Each of these ligands interacts "promiscuously" with many different receptors, and each receptor interacts with many ligands. These promiscuous interactions induce the same change inside the cell but with distinct intensities. Cells recognize complex ligand combinations and thus determine the address of a given message.
Credit: Caltech

A team of Caltech scientists has uncovered a molecular code that cells use to communicate with each other. This "language" is thought to be common to many types of cellular communication and has implications for designing future therapies, according to scientists in the laboratory of Michael Elowitz, professor of biology and bioengineering at Caltech and an investigator with the Howard Hughes Medical Institute.

The work is described in a paper in the September 7 issue of Cell.

Just as humans use letters and words to create messages, cells release molecules called ligands into the environment to send messages to their neighbors—such as an instruction to differentiate or proliferate. The ligands bind to receptors on the surface of other cells, which interpret the message and trigger a set of chemical reactions to relay it to the correct molecules in the cell's interior.

The authors studied a major communication channel, called the bone morphogenetic protein (BMP) pathway, that operates in nearly all tissues and is a target for cancer therapies. This system uses many different ligands and receptors in various combinations. Oddly, the ligands and receptors in this pathway mix and match with each other in many different ways, seemingly for no reason.

"You might think each type of ligand would pair up with only one type of receptor, but this isn't the case," says Elowitz. "Instead, all of the ligands interact promiscuously with all of the receptors. It seems like a big mess, and quite different from the way people design communication systems to keep each communication channel separate. So we wondered why cells communicate in this way."

To figure out what benefit molecular promiscuity provides, the team created different mixtures of BMP ligands in the laboratory and added them to certain cells: breast cells, embryonic stem cells, and connective tissue cells called fibroblasts, all from mice. They found, surprisingly, that the cell decodes information from ligand combinations, not the individual ligands themselves.

"One day, I came to the lab and mixed two ligands together that, each on their own, activate cells very strongly," says Yaron Antebi, a Caltech postdoctoral scholar in biology and biological engineering. "But when I checked the cells the next day, there was no response at all. This led us to theorize that cells aren't listening to individual ligands, but rather to combinations of ligands. It's the combinations that are meaningful. Different combinations of ligands encode different instructions for the cell."

The group also observed that different kinds of cells can extract different information from the same ligand combinations by performing different computations—one cell may add the concentrations of two ligands to receive a message, while another may measure the ratio of the two ligands to decode another message. "We were amazed to find that there is not only one kind of information encoded in these ligands," says James Linton, a research scientist in the Elowitz lab. "Different cells can extract different messages, such as instructions to proliferate or differentiate, from the same molecules."

The group found that this ability to encode multiple messages using the same molecules arises because distinct cell types express different combinations of receptors on their surfaces.

One feature of this communication system is the ability for cells to address messages to specific cell types. "It's kind of like being in a crowded room where everybody is talking, and someone wants to get your attention so they shout your name," says Elowitz. "Everyone else might automatically ignore it, but you pay attention; you are the target of that message."

"This selectivity in communication is extremely useful," adds Antebi. "You can imagine that, if you had a broken bone, you would only want bone cells in the damaged area to proliferate and rebuild the damaged area, not all cells around them."

The findings provide a foundation for understanding how information is distributed across molecules. While the research is at an early stage, a better understanding of cellular communication could lead to designs for biological drugs that could target tumors or particular cell types without affecting any other cells.

The paper is titled "Combinatorial signal perception in the BMP pathway." Other Caltech coauthors include graduate students Heidi Klumpe, Mengsha Gong, Christina Su, and Reed McCardell. The work was funded by the Gordon and Betty Moore Foundation, the Human Frontiers Science Program, the National Institutes of Health, the Defense Advanced Research Projects Agency, and the Institute for Collaborative Biotechnologies.

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